1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 16 NUCLEAR, CYTOPLASMIC, AND MITOCHONDRIAL GLYCOSYLATION Hud Freeze

2. MITOCHONDRIAL GLYCOSYLATION FACT OR FICTION? SUGGESTIVE RESULTS FROM THE EARLY DAYS Suggested by radio labeling with sugar precursors Lectin binding studies SOME NAGGING PROBLEMS Contamination by other cellular components, esp ER How to get glycosyltransferases into mitochondria? How do substrates enter?

3. ER MITOCH CLOSE FRIENDS ??

4. ?

6. GLYCOSYLATION IN MITOCHONDRIA? Metabolic labeling with 35 S +/- deglycosylation 3 H-Mannose labeling

7. Specific mitochondrial protein complexes label with 3 H-Man The glycoprotein binds to The lectin ConA NADH-Ubiquinone-(Complex I) F 1 -ATPase (Complex-V)

8. Nuclear and Cytoplasmic Glycosylation In Favor Many papers Highly regarded journals Many Lectins Composition Metabolic Labeling Enzyme digestions Opposed Orientation of Transferases- Type II Membrane Proteins  Nuclear Localization Signals  No site mapping No structural analysis of glycan Purity

11. CYTOSOLIC GLYCOSYLATION

13. GN Tyr-194 GN Tyr-194 GN Tyr-194 1 2 3 4 GN Tyr-194 5 GN Tyr-194 GN is Self Priming UDP UDP-Glc Phosporylase+ Debranching Enzyme UDP-Glc UDP GS Branching Enz+GS

14. Intersubunit Priming Tyr-194 Intrasubunit Priming Tyr-194

15. CYTOSOLIC GLYCOSYLATION

16. Stages of Dictyostelium Development

17. Skp1 glycosylation pathway in Dictyostelium Most complex form of cytosolic glycosylation

18. Verified Complex Cytosolic Glycosylation Only demonstrated for a single protein, SkpI, in Dictyostelium SkpI involved in ubiquitination of cell cycle proteins Attached O-linked chain at Pro-143 (not conserved) which is first hydroxylated to HyPro Gal  -6Gal  Fuc  1-2Gal  1-3-GlcNA-HyPro GlcNAc and Fuc Transferase purified and shown to have very low Km for sugar nucleotides First Gal and Fuc are added by single transferase Mammalian homologs may not have this modification

19. CYTOSOLIC GLYCOSYLATION

20. O-Mannose UDP-Glc Transfers Glc1-P to Mannose on a cystosolic protein 62kD protein is Phosphoglucomutase (Glc-1-P  Glc-6-P) Transferase and phosphodiesterase found on many cells Membrane association regulated by modification with Glc-1-P Structure of underlying oligosaccharide not known

21. CYTOSOLIC GLYCOSYLATION

25. PROTEOGLYCAN DELIVERY TO NUCLEUS? MAYBE KEY FINDING IS THAT A GAG SUBFRACTION IS HIGHLY ENTRICHED IN ISOLATED NUCLEI MECHANISM?

26. CYTOSOLIC GLYCOSYLATION

27. Ubiquitination of a New Form of  -Synuclein by Parkin from Human Brain: Implications for Parkinson's Disease  Sp22 is an O-linked glycosylated isoform of  -synuclein. Failure of parkin to degrade it could cause some forms of PD SCIENCE, JUNE 28, 2001

28. Accumulation of an O-glycosylated form of  synuclein In autosomal recessive forms of Parkinson’s disease? CYTOSOL

29. co-incubation with O-glycosidase and sialidase A shifted the 22-kD species to a 16-kD position (31), where it now co-migrated with the unmodified S monomer, Sp16, from crude brain extracts (Fig. 5C). We conclude from our data obtained by mass spectrometry analysis (21) and these enzymatic digestions that Sp22 is a posttranslationally modified form of human S containing O-linked sugars. 31. For enzymatic digestion of HP2A precipitates, N-glycosidase, sialidase A, endo-O- glycosidase and protein phosphatase-1 (Sigma) were used as per manufacturers' instructions. 21. The HP2A-specific 22-kD protein yielded tryptic peptides corresponding to aa 13-21, 44-58, 46-58, 59-80, 61-80, 81-96, and 81-97 of human S (GenBank accession # L08850), each ending with a lysine, as expected.  Thr/Ser

30.  Thr/Ser SIALIDASE  Thr/Ser O-GLYCOSIDASE DIGESTION OF O-LINKED GalNAc sugar chain-- 2 easy steps  1. 2.

31. ARE THEY CORRECT OR NOT? WHAT’S YOUR OPINION?